Lab 2 - Protein Quantification Guide F23.pdf

Protein Extraction & Quantification Time to consider what is going on INSIDE the cell! Last lab you observed Ptk2 cell morphology (i.e. how the cells looked) and viability (i.e. the approximate percentage of dead/live cells) in the stressed and unstressed samples. Now we consider how cell biologists...

Protein Extraction & Quantification Time to consider what is going on INSIDE the cell! Last lab you observed Ptk2 cell morphology (i.e. how the cells looked) and viability (i.e. the approximate percentage of dead/live cells) in the stressed and unstressed samples. Now we consider how cell biologists investigate what is happening at a molecular level. There are many approaches to study the inner workings of the cell, but one of the most effective strategies is to lyse the cells and assess protein expression. Techniques like SDS-PAGE and western blotting (which you will do in Labs 3 and 4!) can determine whether a particular protein is up or down-regulated. During the summer, we grew cells in flasks and stressed them with either vinegar or H2O2. We also prepared unstressed cells. After ~15 hr, we extracted the proteins from the cells using RIPA buffer. The resulting cell lysates were aliquoted into many, many, MANY microcentrifuge tubes! We will provide you with cell lysates that correspond to the stressor that you used in Lab 1. You will receive a tube of cell lysate from vinegartreated or H2O2-treated cells, and a tube of cell lysate from unstressed cells. Learning Outcomes: By the time this lab is complete, you should be able to: • • • • • • Prepare a dilution series. Demonstrate pipetting proficiency. Describe how protein can be extracted from cells. Calibrate a spectrophotometer. Construct a standard curve to determine sample concentration. Present data in the form of a table and graph with proper formatting. BEFORE Lab 2, you must: • • • • • Carefully read the rest of this document and print it. (Including Appendix I). Complete the Table 1 calculations below. (yes, complete before the lab!) Complete a flowchart for Lab 2. Complete the pre-lab quiz on Brightspace. (Heads-up, the quiz includes questions from your previous labs!) Plan to bring a laptop (or another device) with you to the lab for constructing a graph. Assessments Related to this Lab: • Following (or during!) your lab session, complete the Lab 2 Assignment (described within this document) and upload the pdf to the assignment folder on Brightspace. It is due by midnight on the day following your lab. McCarville, Garant and Tatar (2023/2024) Background Information: Protein Extraction Cells contain thousands or millions of proteins. If you are interested in protein expression, you must first extract these proteins from your cells. We do this by disrupting the membranes and collecting the resulting liquid supernatant – commonly known as the cell lysate. Proteins can be extracted using physical or chemical lysis methods. In either case, it is critical to make sure that the preparation stays cold, so that the proteins do not denature. Physical methods use external forces to physically break down the membrane to release the cellular components. There are a couple options to extract proteins this way: Liquid Homogenization (e.g. Dounce or Potter-Elvehjem homogenizer) Sonication Manual Grinding (e.g. mortar and pestle) Photo: sigmaaldrich.com Photo: Fishersci.ca Photo: covaris.com This apparatus shears cells by forcing them through a narrow space. This can be done using a plunger and vessel (like shown above) or done in an automated fashion. This machine uses pulsed, high frequency sound waves to agitate and lyse cells. Sound waves are delivered using an apparatus with a vibrating probe that is immersed into the cell suspension. This technique works well to isolate proteins from plant cells. Tissue is frozen in liquid nitrogen and then smashed, releasing the proteins. The challenge with physical methods is that it is often difficult to have consistent preparations from one day to the next. It tends to be less reproducible. The other concern is that these methods can generate heat, which will cause the proteins to denature. Chemical methods use detergents and/or hypotonic solutions (low salt) to disrupt the phospholipid membrane and extract the proteins. RIPA is a commonly used lysis buffer that contains the detergent Triton X-100. RIPA buffer typically includes protease and phosphatase inhibitors. The key steps to extract proteins from adherent cells using RIPA buffer are: • • • • • Grow the cells in flasks. Remove the media and wash the cells with buffer (like PBS) to remove residual media. Add RIPA buffer to the flask, ensuring all cells come in contact with the buffer. Incubate the cells in RIPA buffer on ice for 10+ minutes. Transfer the lysed cells to a tube on ice and centrifuge the contents to pellet any membranes. The supernatant will contain a “soup” of the cell proteins. We refer to this as the cell lysate. McCarville, Garant and Tatar (2023/2024) Background Information: Protein Quantification via a Bradford Assay Once cells have been homogenized, the next step is usually to determine the protein concentration of the cell lysate. Note: Square brackets are often used to indicate the term “concentration”. Normally, we discuss [protein] in units of µg/µl. For example, 0.75 µg/µl means that there is 0.75 µg of protein in 1 µl of the liquid. In 1976, Marion Bradford developed a quick and easy way to find the total concentration of protein in a solution by using a colorimetric reaction. This technique remains one of the most common ways of testing protein concentration! The assay (“assay” is just a fancy word for “test”) is simple; when a dye in the Bradford reagent binds to protein, there is a colour change from brown to blue. The intensity of the blue colour is measured using a spectrophotometer set at 595 nm. This assay uses BSA (bovine serum albumin) as a standard to which the unknown samples will be compared. BSA is isolated from cow blood and is often used in research when a generic protein is needed. Overview of the Bradford Assay: You will start by making a dilution series from stock BSA, which is at a concentration of 1.4 µg/µl. The colour change is linear only in the range of 0.20 – 1.0 µg/µl, therefore the BSA must be diluted with PIPES buffer to make a series of standards in this range. You will prepare 100 µl of each concentration. You will also set up cuvettes for the cell lysates (two cuvettes for the stressed cell sample (“T”) and two cuvettes for the unstressed cell sample (“C”). These samples will also be diluted with buffer. We will provide you with cell lysates that correspond to the stressor that you used in Lab 1. If you previously used vinegar to shock to your cells, we will give you cell lysate from vinegar-treated cells. In contrast, if you tried oxidative stress in Lab 1, we will provide you with a cell lysate from H2O2-treated cells. Once the protein (either BSA or the mixture of proteins from the cell lysate) is diluted in each cuvette, you will add 5 ml of Bradford Reagent to each cuvette. After mixing and allowing a 10-minute incubation, the absorbance (i.e. the “blueness”) is measured using a spectrophotometer. McCarville, Garant and Tatar (2023/2024) Skill #1: Create a serial dilution of the BSA stock solution: 1. BEFORE arriving to the lab, calculate how to prepare your serial dilution of the BSA. Indicate the volumes in Table 1 below. Your stock solution of BSA is at a concentration of 1.4 µg/µl. There are several different ways to make a dilution series. You can use whatever method makes sense to you, but we suggest using the same formula you have used previously (C1V1 = C2V2), where: • C1 = initial concentration of the stock BSA solution (1.4 µg/µl) • V1 = volume of the BSA stock solution (that is what you are solving for) • C2 = the final concentration that you want (e.g., 0.20 µg/µl, 0.30 µg/µl, etc.) • V2 = the final volume that you need (100 µl) Table 1 Cuvette A B C D E F BSA Concentration (µg/µl) Volume of stock BSA (µl) Volume of PIPES buffer (µl) 0 (no protein, this is the blank) 0 100 0.2 0.4 0.6 0.8 1.0 Remember! Use a P20 for volumes 2 - 20 µl; use a P200 for volumes 20 - 200 µl! 2. Label your cuvettes “A” through “J”. Use tape near the top of the tube; do not write directly on the glass! (Note: the cuvettes we use look just like regular test tubes!) 3. Pipette PIPES buffer into the cuvettes. Use your calculated values from Table 1 for cuvettes A-F, and use the values provided below in Step 5 for cuvettes G-J. 4. Pipette BSA stock solution into cuvettes A-F. Use your calculated values from Table 1. You will find the BSA stock solution tube in your ice bucket. Hold the BSA in your gloved hand to thaw it. 5. Pipette cell lysate proteins into cuvettes G-J according to the table below. The volumes are tiny (5 µl – use your P20), so pipette carefully. You will prepare duplicates for each cell lysate sample and take the average. (Remember to put your original tubes containing the cell lysates back on ice!) Cuvette Protein Sample G H I J Negative Control Cell Lysate (“C”) #1 Negative Control Cell Lysate (“C”) #2 Treatment Cell Lysate (“T”) #1 Treatment Cell Lysate (“T”) #2 Volume of PIPES Buffer (µl) 95.0 95.0 95.0 95.0 Volume of Cell Lysate (µl) 5.0 5.0 5.0 5.0 6. Once all 10 cuvettes are prepared, add 5 ml of the Bradford reagent to each cuvette. Stretch Parafilm over the top of each cuvette and invert several times to mix. The reaction will take 10 min to occur. While you wait, proceed to Skill #2: Calibrate the Spectrophotometer McCarville, Garant and Tatar (2023/2024) Skill #2: Calibrate the Spectrophotometer Spectrophotometers use light of a specific wavelength that is generated from suitable light sources. Samples are placed in cuvettes. The type of cuvette can vary depending on the type of spectrophotometer, the volume of the sample, and the wavelength of light. The absorbance of light by the sample is measured by a photocell that converts the received light energy into electrical energy so that a reading is displayed on the unit. The degree to which light is absorbed by a solution (i.e., absorbance) is related to the concentration of the absorbing solution. To account for any absorbance due to the solvent in which the sample is dissolved (i.e., the brown background colour of the Bradford reagent), you will prepare a cuvette that will be used to “blank” the machine by adjusting it to read zero absorbance. The “blank” cuvette will only contain PIPES buffer and the Bradford reagent (no protein!). This ensures that any positive absorbance readings by the spectrophotometer in the remaining cuvettes is the result of the blue colour produced by the Bradford reagent reacting with the proteins. Refer to the instructions on your bench to set up the spectrophotometer. Record the Data 1. Once the 10-minute incubation time is complete, put the Blank (cuvette A) into the spectrophotometer and zero the machine by setting it to 0.00. 2. Record the absorbance values for Cuvettes B-J in Table 2 below. (No need to re-zero the machine between each tube!) Table 2 Absorbance values for all samples. Cuvette Sample A 0 (Blank) B 0.2 µg/µl BSA C 0.4 µg/µl BSA D 0.6 µg/µl BSA E 0.8 µg/µl BSA F 1.00 µg/µl BSA G Negative Control Cell Lysate (“C”) #1 H Negative Control Cell Lysate (“C”) #2 I Treatment Cell Lysate (“T”) #1 J Treatment Cell Lysate (“T”) #2 Absorbance (595 nm) 0.00 McCarville, Garant and Tatar (2023/2024) Skill #3: Construct a Standard Curve to Determine Protein Concentration Use the information from your cuvettes B-F in Table 2 to construct a standard curve (sometimes called a calibration curve) in Excel with BSA concentration on the x-axis and BSA absorbance readings on the y-axis. Do not include the (0, 0) data point of the Blank (i.e. Cuvette A). Make sure to: • • • • Add a linear trendline Set the y-intercept to zero Find the equation of the line and the R-squared value (include this info in your figure caption). Consult the Appendix at the end of this document for presentation expectations. Part of the assessment for this graph will be based on formatting! Determine Cell Lysate Protein Concentrations Yippiee! Now that you have constructed your BSA standard curve, you can determine the protein concentration of your cell lysates! Use the equation of the line generated by Excel to complete the table below. Plug the absorbance values into your equation, and multiply by the dilution factor (remember that we added 5 µl of cell lysate to 95 µl of PIPES buffer – therefore this is a 20X dilution). Cell Lysate Absorbance Reading Protein Concentration in Cuvette (µg/µl) Dilution Factor Negative Control #1 20x Negative Control #2 20x Treatment #1 20x Treatment #2 20x Actual Protein Concentration (µg/µl) *Example 0.740 0.63 20x 12.62 *Example 0.782 0.67 20x 13.33 Actual Protein Concentration (averaged) (µg/µl) 12.98 *Example calculations are shown in the bottom two rows and were performed using y = 1.173x from a BSA standard curve and a 20X dilution (your equation of the line will be different!) Do not round as you go through the different steps of the equation. Only round when you write the values into the final table. Assignment Preparation and Submission: In a separate document that contains your name and your partner’s name, include: 1) The table that indicates your raw data (i.e. absorbance readings for all tubes from Table 2) • Re-label as “Table 1” • No need to format nicely – this table is simply for record keeping and to help your TA grade! 2) Beautifully formatted graph of the BSA Standard Curve • Title it as “Figure 1” and include a caption 3) Beautifully formatted table showing completed calculations of protein concentration. • Label it as “Table 2” and do not include example calculation rows Tables and figures must be prepared electronically with appropriate titles, captions, and formatting. Save it as a pdf, using the file name: Last Name_First Name_F2023_Lab 2 Upload the pdf to the Lab 2 Assignment folder on Brightspace by midnight on the day after your lab. McCarville, Garant and Tatar (2023/2024) Appendix I: Table and Figure Guidelines How to Create a Perfect Biology Table: Title is above the table. There is no colon after the number. • It should be concise (think one long sentence, or two short sentences) • It should be detailed (that does not mean procedural steps). “Detailed” means that the title includes key information that someone would need to understand what the table is about. • No need to include date/time/location (that is specific for Ecology) • Never start the title by saying “Table showing…” Lines • Thick line above and below the table. Thin lines below the header row. • No lines anywhere else • Tip: you can change the line thickness in Word by going under Table Design and using “Borders & Shading”, the available quick keys, or “Border Painter”. Columns • Do not include a column that lists “tube #” or “sample #” if it refers to randomly numbered tubes or samples that were used to set up the experiment. Data • • If the value is a volume (e.g., µl) or a concentration, indicate two decimal places. You cannot transfer volumes smaller than two decimal places using a micropipettor. When doing a multi-step calculation, do not round as you go through the different steps of the actual equation. Round only the very final answer. (However, if you are writing data into a table, you will have to insert the number rounded to two decimal places.) Example below! (Notice how this table is clean and uncluttered! So pretty!) Table 1 Average flagellar length measurements for Chlamydomonas cultures in regular media and in cycloheximide over an 80-minute time trial. Flagella were measured with an Olympus microscope using phase contrast at 400x magnification. Sampling Time (min) Regular Media (μm) Cycloheximide (μm) 0 0.65 0.25 20 8.25 4.56 60 10.53 6.68 McCarville, Garant and Tatar (2023/2024) How to Present a Perfect Graph: Caption is below the figure. Again, no colon after the number. • Figure captions vary in length, but aim for ~2 sentences. • The caption should be concise and detailed (that does not mean procedural steps). “Detailed” means that the caption includes key information that someone would need to understand what the figure is about without reading the rest of the document. o What variable is being measured? How is the variable being measured? o Include the equation of the line and the R2 value. o Do not include date/time/location (that is specific for Ecology) o Never start the caption by saying “Graph of…” or “Figure of…” • Only the caption below is required. There should not be a chart title above the table. Lines • No gridlines on the graph. • No border. • The plot area (background) should be white. Data • If there are multiple plotlines, you should include a key or legend. If there is only one plotline, do NOT include a key or legend. • Tip: add a linear trendline in Excel by right-clicking on a data point and selecting “add trendline”. Example below! (Again, black and white, clean and uncluttered!) Figure 1 Fermentation in Saccharomyces cerevisiae was measured by CO2 production. Various glucose concentrations (2-10%) were used and volume of CO2 was determined by measuring the changes in fluid volumes (in ml) within the reaction tube. The equation of the line is y = 0.4318x + 1.02 and R2 = 0.975. The assignment is due by midnight on the day after your lab. (e.g. if your lab is on Wednesday, your assignment must be submitted by midnight on Thursday!) It is to be submitted individually. However, similarities between your assignment and your partner’s assignment are to be expected and are not a problem. 😊 McCarville, Garant and Tatar (2023/2024)